Enkephalin derivatives

ABSTRACT

Improved enkephalin derivatives represented by the formula: ##STR1## wherein: X is a chiral residue of a D-amino acid selected from the group consisting of D-alanine, D-leucine, D-isoleucine, D-valine, D-norvaline, D-phenylalanine, D-tyrosine, D-tryptophan, D-serine, D-threonine, D-methionine, D-glutamic acid, D-glutamine, D-aspartic acid, D-asparagine, D-lysine, D-proline, D-histidine and D-arginine; Y is a residue of methionine or leucine and Z is selected from the group consisting of hydroxy, amino, loweralkylamino, diloweralkylamino and loweralkoxy; and the pharmaceutically acceptable salts thereof; intermediates useful in making the novel pentapeptides and pharmaceutical compositions and methods employing them.

BACKGROUND OF THE INVENTION

While there are a number of analgesic agents currently utilized torelieve mild to severe pain, the search for improved analgesics is acontinuing one because of the numerous problems associated with thepresently available agents. Aspirin and related salicylates areconsidered to be non-narcotic analgesic agents useful for relieving mildto moderate pain in addition to their usefulness as anti-inflammatoryand anti-pyretic agents. However, the ingestion of salicylic acid orrelated salicylates may result in epigastric distress, nausea andvomiting. This widely used class of non-narcotic analgesic agents mayalso cause gastric ulceration and even hemorrage both in experimentalanimals and man. Exacerbation of peptic ulcer symptoms and erosivegastritis have all been reported in patients on high dose therapy, i.e.,arthritis patients. Aspirin is also one of the most common causes ofdrug poisoning in young children and has a potential of serious toxicityif used improperly.

Acetaminophen is also considered to be a non-narcotic analgesic agentuseful in treating mild pain associated with simple headache, commonmuscular aches, etc. While acetaminophen is particularly useful forpatients who cannot take aspirin, i.e., ulcer patients, its use iscontraindicated in individuals who have exhibited a sensitivity to it.In addition to their drawbacks in view of their potential side effects,the mild non-narcotic analgesic agents are not sufficiently potent torelieve the severe pain associated with surgery, cancer and the like.

Unfortunately, the potent analgesic agents capable of relieving suchsevere pain are also narcotic agents and their use entails the risk ofproducing physical and sometimes psychological dependence. There are asyet no agents effective against severe pain that are entirely free ofthis risk.

Thus, there is an urgent need for improved analgesic agents for treatingmild as well as severe pain. The present invention provides such agents.

In addition to the need for improved analgesic agents, there is also aneed for improved psychotropic agents to replace or to provide analternative to current therapy. The compounds of this invention, inaddition to their analgesic activity, also exhibit anti-depressantactivity. Thus their usefulness as analgesic agents is enhanced sincemany patients suffering from pain also exhibit varying states of anxietyand depression.

In 1975, Hughes et al. identified a pentapeptide, methionine enkephalin,which has the following structure, H-Tyr-Gly-Gly-Phe-Met-OH [see Hugheset al., Nature, 258, 577 (1975)]. This peptide is found in many areas ofthe brain where it appears to act as a neurotransmitter orneuromodulator in a central pain suppressive system. The natural peptidebinds stereospecifically to partially purified brain opiate receptorsites [for instance see Bradbury et al., Nature, 260, 793 (1976)], isvery active in bioassays for opiate activity, but exhibits only weakanalgesic activity of short duration when injected directly into thebrain of the rat, [for instance, see Belluzzi et al., Nature, 260, 625(1976)].

We subsequently found that when methionine enkephalin was substituted inthe 2-position with a D-amino acid, potent analgesic agents areobtained. The D-amino acid² analogs also exhibit other central nervoussystem and hormonal activities.

We have now found that even greater in vivo analgesia is obtained whenthe D-amino acid² enkephalins are additionally substituted in the4-position by a pentafluorophenylalanine

SUMMARY OF THE INVENTION

This invention relates to novel pentapeptides, and more specificallyrelates to improved methionine and leucine enkephalin derivatives whichare useful as analgesic and anti-depressant agents, tranquilizers,sedative-hypnotics, growth hormone promoters and prolactin releasingagents, to intermediates useful in the preparation of the novelpentapeptides, and to pharmaceutical compositions and methods employingsuch novel pentapeptides.

Specifically, the novel pentapeptides are methionine or leucine2-D-amino acid-4-pentafluorophenylalanine enkephalins and derivativesthereof.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The compounds of this invention are represented by formula I: ##STR2##wherein x is a chiral residue of a D-amino acid selected from the groupconsisting of D-alanine, D-leucine, D-isoleucine, D-valine, D-norvaline,D-phenylalanine, D-tyrosine, D-tryptophan, D-serine, D-threonine,D-methionine, D-glutamic acid, D-glutamine, D-aspartic acid,D-asparagine, D-lysine, D-proline, D-histidine and D-arginine; and Y isa residue of methionine or leucine and Z is selected from the groupconsisting of hydroxy, amino, loweralkylamino, diloweralkylamino andlower alkoxy; and the pharmaceutically acceptable salts thereof.

The term "pharmaceutically acceptable salts", as used herein, refers tothe non-toxic alkali metal, alkaline earth metal and ammonium saltscommonly used in the pharmaceutical industry including the sodium,potassium, lithium, calcium, magnesium, barium and ammonium salts whichare prepared by methods well known in the art. The term also includesnon-toxic acid addition salts which are generally prepared by reactingthe compounds of this invention with a suitable organic or inorganicacid. Representative salts include the hydrochloride, hydrobromide,sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, napsylate and the like.

The term "lower alkyl" refers to straight and branched chain alkylgroups having from 1 to 6 carbon atoms such as methyl, ethyl, n-propyl,isopropyl, n-butyl, ser-butyl, tert-butyl, n-pentyl, n-butyl and thelike.

All chiral amino acid residues identified herein are in the natural orL-configuration unless otherwise specified. In keeping with standardpeptide nomenclature, abbreviations for chiral amino acid residues havebeen used herein as follows:

    ______________________________________                                        His                                                                           L-histidine            D-His                                                  D-histidine                                                                   Tyr                                                                           L-tyrosine                                                                          Ile                                                                     L-isoleucine                                                                  D-Tyr                                                                         D-tyrosine                                                                          D-Ile                                                                   D-isoleucine                                                                  Gly                                                                           glycine                                                                             Leu                                                                     L-leucine                                                                     Phe                                                                           L-phenylalanine                                                                     D-Leu                                                                   D-leucine                                                                     D-Phe                                                                         D-phenylalanine                                                                     Thr                                                                     L-threonine                                                                   Met                                                                           L-methionine                                                                        D-Thr                                                                   D-threonine                                                                   D-Met                                                                         D-methionine                                                                        Val                                                                     L-valine                                                                      Ala                                                                           L-alanine                                                                           D-Val                                                                   D-valine                                                                      D-Ala                                                                         D-alanine                                                                           Pro                                                                     L-proline                                                                     Ser                                                                           L-serine                                                                            D-Pro                                                                   D-proline                                                                     D-Ser                                                                         D-serine                                                                            Gln                                                                     L-glutamine                                                                   Lys                                                                           L-lysine                                                                            D-Gln                                                                   D-glutamine                                                                   D-Lys                                                                         D-lysine                                                                            Glu                                                                     L-glutamic acid                                                               Asn                                                                           L-asparagine                                                                        D-Glu                                                                   D-glutamic acid                                                               D-Asn                                                                         D-asparagine                                                                        Trp                                                                     L-tryptophan                                                                  D-Asp                                                                         D-aspartic acid                                                                     D-Trp                                                                   D-tryptophan, etc.                                                            ______________________________________                                    

Also contemplated within the scope of the present invention areintermediates of formula II:

    R.sup.3 -Tyr(R.sup.2)-X-Gly-Phe-Y-R.sup.1                  (II)

wherein

R¹ is selected from a group consisting of NH₂, OH, or a derivatizedinsoluble polystyrene resin support represented by the formulae III orIV as follows: ##STR3## R² is a protecting group for the phenolichydroxyl group of tyrosine selected from the group consisting oftetrahydropyranyl, tert-butyl, trityl, benzoyl, 2,4-dichlorobenzyl,benzyloxycarbonyl, and 2-bromobenzyloxycarbonyl (2-Br-Z).

R₃ is a protecting group which would be used by one skilled in the artof solid-phase synthesis of the peptides (I) selected from the groupconsisting of acyl type protecting groups, aromatic urethan-typeprotecting groups, cycloalkyl urethan protecting groups, thio urethantype protecting groups, alkyl type protecting groups, trialkylsilanegroups or aliphatic urethan protecting groups; and

X is a D-amino acid as defined in formula (I) or, when X is D-tyrosine,D-threonine, D-serine, D-glutamic acid or D-lysine, a protected chiralamino acid residue as defined below.

When X is, D-tyrosine; the protected chiral residue is X(R₂) wherein R²is as described hereinabove.

When X is D-serine or D-threonine, the protected residue is X(R₂)wherein R₂ is a protecting group for the alcoholic hydroxyl function andis as defined above.

When X is D-glutamic acid, D-aspartic acid, the protected residue isX(R₄) wherein R₄ is benzyl or tertbutyl. In the case of D-lysine, theprotected residue is X (R₅) wherein R₅ is a protecting group for theepsilon amino function selected from the group consisting ofbenzyloxycarbonyl or 2-chlorobenzyloxycarbonyl.

When X is as D-arginine, the protected residue is X(R₆) wherein R₆ is aprotecting group for the guanidino function such as tosyl or nitro.

The term "acyl type protecting groups" refers to groups illustrated butnot restricted to formyl, trifluoroacetyl, tosyl, nitrosulfonyl, and thelike.

The term "aromatic urethan-type protecting groups" is represented bygroups such as benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-biphenylisopropyloxycarbonyl, 2,5-dimethoxyphenyl isopropyloxycarbonyl, and thelike.

The term "cycloalkyl urethane protecting group", as used herein, refersto groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl,cyclohexylcarbonyl, isobornyloxycarbonyl, etc.

"Urethane type protecting groups" include but are not limited to groupssuch as phenylthiocarbonyl.

"Alkyl type protecting groups" are those commonly used in the art suchas trityl.

"Trialkysilane groups" include compounds such as trimethylsilane,triethylsilane, tributylsilane, and the like.

The preferred protecting groups, the "aliphatic urethane protectinggroups" include tert-butyloxycarbonyl, diisopropyloxycarbonyl,isopropyloxycarbonyl, allyloxycarbonyl and the like.

The polystyrene resin support is preferably a copolymer of styrene withabout 1-2% divinyl benzene as a cross-linking agent which causes thepolystyrene polymer to be completely insoluble in most organic solvents.In formula III, φ is phenyl.

In selecting a particular side-chain protecting group to be used in thesynthesis of the peptides of formula I, several conditions must be met:(a) the protecting group must be stable to the reagent and underreaction conditions selected for removing the α-amino protecting groupat each step of the synthesis; (b) the protecting group must retain itsprotecting properties and not be chemically modified; and (c) theside-chain protecting group must be removable at the end of thesolid-phase synthesis under reaction conditions that will not alter thepeptide chain.

The pentapeptides of formulae (I) and (II) are prepared using standardsolid-phase techniques. The synthesis is commenced from the C-terminalend of the peptide using an α-amino protected resin. A suitable startingmaterial can be prepared, for instance, by attaching an α-aminoprotected methionine to a chloromethylated resin, a hydroxymethyl resin,or a benzyhydrylamine resin. One such chloromethylated resin is soldunder the tradename Bio-beads SX-1 by Bio Rad Laboratories, Richmond,California and the preparation of the hydroxymethyl resin is describedby Bodanszky et al., Chem. Ind.(London) 38, 1597 (1966). Thebenzyhydrylamine resin has been described by Pietta and Marshall, Chem.Commun., 650 (1970) and is commercially available from BeckmanInstrument, Palo Alto, California.

In the preparation of the compounds of this invention, α-aminoprotected-methionine or leucine is coupled to the chloromethylated resinwith the aid of, for example, cesium bicarbonate catalyst, according tothe method described by Gisin, Helv, Chim. Acta, 56, 1476 (1973). Afterthe initial coupling, the α-amino protecting group is removed by achoice of reagents including trifluoroacetic acid or hydrochloric acidsolutions in organic solvents at room temperature. After removal of theα-amino protecting group, the remaining protected amino acids arecoupled stepwise in the desired order to obtain the compounds of formulaII. Each protected amino acid is generally reacted in a 3-fold excessusing an appropriate carboxyl group activator such asdicyclohexylcarbodiimide in solution in, for example, methylenechloride-dimethylformamide mixtures.

After the desired amino acid sequence has been completed, the desiredpeptide is removed from the resin support by treatment with a reagentsuch as hydrogen fluoride which not only cleaves the peptide from theresin, but also cleaves all remaining side-chain protecting groups. Whenthe chloromethylated resin is used, hydrogen fluoride treatment resultsin the formation of the free peptide acids of formula I (Y═0H). When thebenzhydrylamine resin is used, hydrogen fluoride treatment resultsdirectly in the free peptide amides of formula I (Y═NH₂). Alternatively,when the chloromethylated resin is employed, the side-chain protectedpeptide can be cleaved by treatment of the peptide-resin with ammonia togive the desired side-chain protected amide or with an alkylamine ordialkylamine to give a side-chain protected alkylamide or dialkylamide.Side-chain protection is then removed in the usual fashion by treatmentwith hydrogen fluoride to give the amides of formula I.

In preparing the esters of this invention (Z═lower alkoxy), the resinused to prepare the acids of formula I (Y═OH) is employed and thepeptide is removed from the resin by treatment with base and theappropriate alcohol, i.e., methanol. Side chain protection is thenremoved in the usual fashion by treatment with hydrogen chloride toobtain the desired ester.

The solid-phase procedure discussed above is well known in the art andhas been essentially described by J. M. Stewart, "Solid Phase PeptideSynthesis:" (Freeman and Co., San Francisco, 1969).

The compounds of formula I are useful as analgesic and anti-depressantagents when administered to mammalian hosts at dosages of from 0.0001 to5 mg/kg of body weight daily, preferably in divided dosages. Thecompounds are preferably administered by parenteral routes, i.e., theintravenous, intraperitoneal, intramuscular or subcutaneous routes ofadministration. The compounds may also be administered by a variety ofother routes including oral or sublingual, vaginal, rectal or nasalroutes of administration. Accordingly, one aspect of the presentinvention includes pharmaceutical compositions suitable for such routesof administration.

The analgesic activity of the compounds of formula I is established inthe rat tail flick test as described by D'Amour and Smith, J. Pharmac.Exp. Ther., 72, 74 (1941).

The anti-depressant tranquilizing and sedative hypnotic activities areestablished in the open field test described by Kullarni et al.Pharmakopsychiatrie News Psychopharmakologie 8(1): pp 45-50 (1975) andthe self stimulation test described by Bailey et al., ResearchCommunications in Chemical Pathology and Pharmacology 11 (4): pp 543-552(1975). The release of radioimmuno assayable growth hormone andprolactin release after intraceroventribular injection in rats was alsoassayed.

The following examples further illustrate the present invention.

EXAMPLE 1 Preparation of-02-Bromobenzyloxycarbonyl-L-tyrosyl-D-alanyl-glycyl-L-pentafluorophenylalanyl-L-methionyl-benzhydrylamineresin

Benzyhydrylamine resin (0.94 g 0.50 mmole), (purchased from BeckmanInstruments, Palo Alto, California), was placed in the reaction vesselof a Beckman Model 990 automatic peptide synthesizer, programmed tocarry out the following cycle of washes and reactions: (a) methylenechloride; (b) 33% trifluoroacetic acid in methylene chloride (2 timesfor 2.5 and 25 minutes each); (c) methylene chloride; (d) ethanol; (e)chloroform; (f) 10% triethylamine in chloroform (2 times for 5 minuteseach); (g) chloroform; and (h) methylene chloride.

The free amino resin was then stirred with tertbutyloxycarbonyl (t-Boc)methionine (347 mg., 1.5 mmoles) in methylene chloride, anddicyclohexylcarbodiimide (1.5 mmoles) was added thereto. The mixture wasstirred at room temperature for 2 hours and the peptide resin was thenwashed successively with methylene chloride (3 times), ethanol (3times), and methylene chloride (3 times). The attached amino acid wasdeprotected with 33% trifluoroacetic acid in methylene chloride (2 timesfor 2.5 and 25 minutes each) and then steps (c) through (h) as describedin the above wash were performed.

The following amino acids (1.5 mmoles) were then coupled successively bythe same cycle of events; t-Boc-pentafluorophenylalanine t-Boc-Gly;t-Boc-D-Ala; t-Boc-Tyr (2-Br-Z). The completed pentapeptide resin waswashed with methanol (3 times) and dried under reduced pressurewhereupon 1.26 g of material was obtained.

EXAMPLE 2 Preparation of L-tyrosyl-D-alanyl-L-glycyl-L-pentafluorophenylalanyl-L-methionine enkephalin amide

Removal of the protecting group and cleavage of the pentapeptide fromthe resin obtained according to the method of Example 1 was carried outby treating 1.26 g of the peptide-resin with hydrogen fluoride (20 ml)and anisole (2 ml) at 0° C. for 45 minutes. The hydrogen fluoride wasremoved at reduced pressure and the anisole removed by washing withethyl acetate.

The crude peptide was purified by gel filtration on a column (2.5×95 cm)of Sephadex G-15 by elution with 0.2 molar acetic acid and fractionsshown to contain a major peak by uv absorption at 280 nm were pooled andevaporated to dryness.

The residual oil was applied to a column (2.5×95 cm) of Sephadex G-25,previously equilibrated with the lower phase followed by the upper phaseof n-butanol: acetic acid: water (4:1:5) solvent system. Elution withthe upper phase yielded a major symmetrical peak and material from thiswas evaporated to dryness and lyophilized from dilute acetic acidsolution to yield a white, fluffy powder (45 mg).

The product was homogeneous by thin layer chromatography in fourseparate solvent systems on silica gel when loads of 20 ug were appliedand visualized by exposure to ninhydrin reagent followed bychlorine/starch-potassium iodide reagent. The following Rf values wereobtained:

(A) 1-butanol: acetic acid: water (4:1:5 upperphase), 0.58; (B) ethylacetate: pyridine: acetic acid: water (5:5:1:3) 0.84; (C) 2-propanol: 1M acetic acid (2:1), 0.70; (D) 1-butanol: acetic acid: water: ethylacetate (1:1:1:1), 0.68. Amino acid analysis gave: Gly: 1.00; Ala, 0.98;Met + pentafluorophenylalanine, 2.02; Tyr, 0.99.

EXAMPLE 3 Preparation of0-2-Bromobenzyloxycarbonyl-L-tryrosyl-D-leucyl-L-glycyl-L-pentafluorophenylalanyl-L-methionyl-benzhydrylamineresin

Using the conditions described in Example 1, the t-Boc derivatives ofpentafluorophenylalanine, glycine, D-leucine, and tyrosine (2-Br-Z) arecoupled successively to a methionine benzhydryl resin to produce thedesired resin.

EXAMPLE 4 Preparation ofL-tyrosyl-D-leucyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

The above-named compound is prepared by deprotecting and cleaving thedesired peptide from the resin support of Example 3 under the conditionsdescribed in Example 2. The crude peptide is purified by gel filtrationon a column (2.5×95 cm) of Sephadex G-15 by elution with 2.0 molaracetic acid followed by partition chromatography as described in Example2.

EXAMPLE 5 Preparation of0-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-phenylalanyl-L-pentafluorophenylalanyl-L-methionyl-benzhydrylamineresin

Using the conditions described under Example 1, the t-Boc derivatives ofpentafluorophenylalanine, glycine, D-phenylalanine and tyrosine [Phe,Gly, D-Phe, and Tyr (2-Br-Z)] are coupled successively to amethionine-benzhydryl resin (0.63 g, 0.50 mmole) to yield the desiredpeptide resin.

EXAMPLE 6 Preparation ofL-tyrosyl-D-phenylalanyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

The pentapeptide is deprotected and cleaved from the resin support ofExample 5 under the conditions described in Example 2. The crude peptideis purified by gel filtration on a column (2.5×95 cm) of Sephadex G-15by elution with 50% acetic acid followed by partition chromatography asdescribed in Example 2.

EXAMPLE 7 Preparation of0-2-bromobenzyloxycarbonyl-L-tyrosyl-D-alanyl-L-pentafluorophenylalanyl-L-methionyl-O-CH2-resin

Using the conditions described under Example 1, the t-Boc derivatives ofpentafluorophenylalanine, glycine, D-alanine and tyrosine (2-Br-Z) werecoupled to tertbutyloxycarbonyl-me thionine-O-CH₂ -resin (0.5 mmole)prepared by the method of Gisin, Helv. Chim. Acta., 56, 1476 (1973) toobtain the desired resin.

EXAMPLE 8 Preparation ofL-tyrosyl-D-alanyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

The above pentapeptide resin was deprotected and cleaved from the resinsupport of Example 7 under the conditions described in Example 2. Thecrude material was purified by gel filtration and partitionchromatography as described in Example 2 to yield the desired product.

The following compounds are illustrative of additional compounds offormula I which are prepared according to the methods of Examples 1-8:

L-Tyrosyl-D-isoleucyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-tyroyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-tryptophyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-serinyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-threonyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-methionyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-glutamyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-aspartyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-asparaginyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-lysyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-arginyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-glutaminyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin

L-Tyrosyl-D-isoleucyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-tyrosyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-tryptophyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-serinyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-threonyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-methionyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-glutamyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-aspartyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-asparginyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-lysyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-arginyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-glutaminyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin amide

L-Tyrosyl-D-isoleucyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-tryptophyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-serinyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-threonyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-methionyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-glutamyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-aspartyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-asparaginyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-lysyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-arginyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-glutaminyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin

L-Tyrosyl-D-isoleucyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-tyrosyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-tryptophyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-serinyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-threonyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-methionyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-glutamyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-aspartyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-asparginyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-lysyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tyrosyl-D-arginyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide

L-Tryosyl-D-glutaminyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide and salts of the above compounds.

The present invention includes within its scope pharmaceuticalcompositions comprising, as an active ingredient, at least one of thecompounds of formula I in association with a pharmaceutical carrier ordiluent. The compounds of this invention can be administered by oral,parenteral, nasal, vaginal, rectal or sublingual routes ofadministration and can be formulated in dosage forms appropriate foreach route of administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert diluent such as sucrose,lactose, or starch. Such dosage forms can also comprise, as is normalpractice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water.Besides, inert diluents, such compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil, andinjectable organic esters such as ethyl oleate. Such dosage forms mayalso contain adjuvants such as preserving, wetting, emulsifying, anddispersing agents. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, by irradiating the compositions, or by heatingthe compositions. They can also be manufactured in the form of sterilesolid compositions which can be dissolved in sterile water, or someother sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax.

Compositions for nasal or sublingual administration are also preparedwith standard excipients well known in the art.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment.Generally, dosage levels of between 0.0001 to 5 mg./kg. of body weightdaily are administered to mammals to obtain effective relief from painor to relieve depression.

The following examples further illustrate the pharmaceuticalcompositions which are a feature of this invention.

EXAMPLE 9

Tablets weighing 200 mg. and having the following compositions areformulated:

    ______________________________________                                        Ingredient            Mg                                                      ______________________________________                                        L-Tyrosyl-D-alanyl-L-glycyl-                                                  L-pentafluorophenylalanyl-L-                                                  methionine enkephalin amide                                                                         25                                                      Starch                145                                                     Colloidal silica      27                                                      Magnesium stearate     3                                                      ______________________________________                                    

EXAMPLE 10

Sterile 10 ml. ampoules can be prepared containing 1 mg per ml ofL-tyrosyl-D-alanyl-L-glycyl-L-pentafluorophenylalanyl-L-methionineenkephalin, 0.1 percent sodium bisulfate, 0.7 percent sodium chloride,and 0.5 percent chlorobutanol as a preservative.

EXAMPLE 11

Topical aqueous formulations for administration by nose drops or nasalspray are formulated containing 1 mg ofL-tyrosyl-D-leucyl-L-glycyl-L-pentafluorophenylalanyl-L-leucineenkephalin amide, 3.8 mg. glycerine, 40 mg. sorbital, 0.02 mg.benzalkonium chloride and purified water q.s. 1 ml.

We claim:
 1. A pentapeptide of the formula ##STR4## wherein: X is achiral residue of a D-amino acid selected from the group consisting ofD-alanine, D-leucine, D-isoleucine, D-valine, D-norvaline,D-phenylalanine, D-tyrosine, D-tryptophan, D-serine, D-threonine,D-methionine, D-glutamic acid, D-glutamine, D-aspartic acid,D-asparagine, D-lysine, D-proline, D-histidine and D-arginine; Y ismethionine or leucine and Z is selected from the group consisting ofhydroxy, amino, loweralkylamino, diloweralkylamino and loweralkoxy; andthe pharmaceutically acceptable salts thereof.
 2. A compound of claim 1wherein Y is methionine.
 3. A compound of claim 2 wherein Z is hydroxy.4. A compound of claim 2 wherein Z is amino.
 5. A compound of claim 2wherein Z is loweralkylamino.
 6. A compound of claim 2 wherein Z isdiloweralkylamino.
 7. A compound of claim 1 wherein Z is loweralkoxy. 8.A compound of claim 4: D-Ala² -F₅ Phe⁴ -methionine enkephalin amide or apharmaceutically acceptable salt thereof.
 9. A compound of claim 1wherein Y is leucine.
 10. A compound of claim 9 wherein X is hydroxy.11. A compound of claim 10 wherein X is amino.
 12. A compound of claim10 wherein X is loweralkylamino.
 13. A compound of claim 10 wherein X isdiloweralkylamino.
 14. A compound of claim 10 wherein X is loweralkoxy.15. A pharmaceutical composition comprising administering an effectiveamount of a compound of claim 1 and a pharmaceutically acceptablecarrier or diluent.